Ab199013

Immunostaining for tumor necrosis factor-α (TNF-α), RANKL and osteoprotegerin (OPG) was performed on 4-μm decalcified right distal femoral sections. Briefly, all sections were deparaffinized in xylene, rehydrated, and washed in PBS. The sections were then heated for 20 min in citrate buffer (pH 6.0) at 95°C and incubated with an anti-tumor necrosis factor-α (TNF-α) monoclonal antibody (1:100, catalog number: ab199013, Abcam, United States), anti-RANKL polyclonal antibody (1:200, catalog number: BA1323, Wuhan Boster, China), and anti-Osteoprotegerin (OPG) polyclonal antibody (1:500, catalog number: ab73400, Abcam, United States) overnight at 4°C. Phosphate-buffered saline (PBS) was used as a negative control. Then, the sections were incubated with secondary antibodies for 30 min at 26°C. 3,3′-Diaminobenzidine (DAB) staining and hematoxylin counterstaining were performed to visualize the expression of biomarkers. Immunohistochemical analysis was performed with Image-Pro Plus 6.0 software to evaluate immunostaining.

The eyes were harvested at defined times after RD. The animals were euthanized, and the eyes were enucleated. The whole eyes were fixed with 4% paraformaldehyde (PFA) solution in phosphate-buffered saline (PBS) for 24 h at 4 °C, dehydrated in 30% sucrose solution for 24 h at 4 °C, embedded in OCT at −20 °C, and sectioned at 10 μm using a Leica cryostat. Eye sections were fixed in 4% PFA/PBS for 10 min, washed with PBS twice and blocked for 1 h in 5% BSA in PBS containing 0.1% Triton-X (PBST) at room temperature. The primary antibodies were diluted in antibody dilution solution (1% BSA in PBST) in ratios from 1:100 to 1:1000 and used for incubation overnight at 4 °C. The secondary antibodies were diluted at 1:1000 in antibody dilution solution and applied for incubation for 2 h at room temperature. The primary antibodies used for immunofluorescence were LC3B (Cell Signaling Technology, 2775S, 1/1000), Atg5 (Abcam, ab78073, 1/100), and TNF-α (Abcam, ab199013, 1/100). The secondary antibodies used were Cy3 donkey anti-mouse IgG, Cy3 donkey anti-rabbit IgG (Jackson ImmunoResearch, 715-165-150, 711-165-152), Alexa488 donkey anti-mouse IgG, and Alexa488 donkey anti-rabbit IgG (Life Technologies, A16017, A16033). All images were obtained using a Zeiss LSM 780 confocal microscope. In each experiment, measurements were made on three eyes.

IL-8, TNF-α, CXCL5 and β-actin mRNA levels were determined by quantitative reverse transcription PCR (qRT-PCR)[21 (link)]. The primers used were as follows: IL-8 forward primer (5’- ATG ACT TCC AAG CTG GCC GTG GCT-3’) and reverse primer (5’-TCT CAG CCC TCT TCA AAA ACT TCT C -3’); TNF-α forward primer (5’- CGA GTG ACA AGC CTG TAG C -3’) and reverse primer (5’-CCT TCT CCA GCT GGA GAG C-3’); CXCL5 forward primer (5’-GAG AGC TGC GTT GCG TTT G-3’) and reverse primer (5’-TTT CCT TGT TTC CAC CGT CCA-3’); GAPDH forward primer (5’-ACCACAGTCCATGCCATCAC-3’) and reverse primer (5’-TCCACCACCCTGTTGCTGTA-3’). Sample input was normalized to β-actin mRNA expression. IL-8, TNF-α, CXCL5 and β-actin protein levels were determined by western blot analysis[22 (link)], using following antibodies: ab34100 (1:1000 dilution, abcam, Cambridge, MA) for IL-8, ab199013 (1:1000 dilution, abcam, Cambridge, MA) for TNF-α, ENA-78 (1:1000 dilution, Santa Cruz Biotechnology, Santa Cruz, CA) for CXCL5 and ab8226 (1:500 dilution, abcam, Cambridge, MA) for β-actin.

Liver tissue sections were dewaxed, hydrated, and washed. After neutralization with 3% endogenous peroxidase and microwave antigen retrieval, slides were preincubated with blocking serum and then incubated overnight with antibodies against tumor necrosis factor (TNF)-α (ab199013, dilution 1:200), interleukin (IL)-6 (ab9324, dilution 1:200), and hepatocyte growth factor (HGF) (ab83760, dilutions 1:200) (Abcam). Three images of 5 representative fields were captured using a Canon EOS600D camera connected to a microscope at a magnification of × 200. Images were analyzed with Image-Pro Plus version 6.2 software (Media Cybernetics), using a special function called “measurement of integrated absorbance”. The integrated absorbance of positive staining of TNF-ɑ, IL-6, and HGF in each photograph was measured, and its ratio to total area of each photograph was calculated as density. The average integrated absorbance value (integrated absorbance/total area) of each slide (3 images) was used to represent a particular sample.